Project Description. Trypanosomatid parasites are the causative agents of human African trypanosomiasis (HAT), Leishmaniasis and Chagas disease, all of which are listed by the WHO as Neglected tropical diseases (NTDs). Collectively 18-20 million people are infected with one of these parasites, yet drug therapies remain inadequate for all three diseases. Discovery of a drug-target that could be exploited against all three parasites, while not showing toxicity against the host, would provide a robust new approach to combat these diseases. Eukaryotes universally require a translation factor called eIF5A for cell growth. To be active eIF5A must be post-translationally modified on a conserved lysine reside to generate a novel amino acid called hypusine. The hypusine modification of eIF5A is catalyzed by deoxyhypusine synthase (DHS) using spermidine as a substrate. DHS is an essential enzyme in all eukaryotes including the trypanosomatids. We previously demonstrated that DHS from trypanosomatids and Entamoeba have uniquely evolved to require heterotetramer formation between two paralogous gene products to generate the active enzyme. This unusual configuration of these parasite DHSs suggests that it may be feasible to identify selective inhibitors of the parasite enzymes that don't inhibit human DHS. The goal of this proposal is to determine if drug-like inhibitors of DHS that are selective for the trypanosomatid enzymes can be identified and if so to advance them during a lead optimization program for the treatment of HAT. In the R21 phase of this proposal we plan to develop a high throughput screen (HTS) compatible assay for T. brucei DHS and to use this assay to screen the UT Southwestern 200,000 compound library for T. brucei DHS inhibitors. We will perform hit validation on identified inhibitors and determine if the identified inhibitors show good selectivity versus human DHS. If trypanosomatid specific DHS inhibitors with good drug like properties can be identified than in the R33 phase of the proposal we plan a hit to lead optimization of at least one series with appropriate properties to be advanced for the treatment of HAT. Identified DHS inhibitors will also be tested at various stages during hit validation and lead optimization against T. cruzi and Leishmania to determine if they have the potential to be developed as a broad-spectrum treatment for all three trypanosomatid-based diseases.